(1) Field of the Invention
The present invention relates to the field of molecular biology, and recombinant bioengineering. In particular, the invention relates to materials and methods for integrating heterologous nucleic acids into the genome of a host organism with little or no disturbance of expression of the genes located in the integration site of the host.
(2) Description of Related Art
Recombinant bioengineering technology has enabled the ability to introduce heterologous or foreign genes into hosts and organisms to produce hosts and organisms that can then be used for the production and isolation of the proteins encoded by the heterologous genes. Numerous recombinant expression systems are available for expressing heterologous genes in mammalian cell culture, plant and insect cell culture, and microorganisms such as yeast and bacteria. However, currently available recombinant expression systems are subject to factors that can limit their utility. These factors include the stability of the transformants; the viability of the recombinantly transformed vector and hosts; and the ability to control for factors such as number of copies of the plasmid vector incorporated into a host genome.
Current methods of recombinant bioengineering for producing stable recombinant hosts commonly use so-called “knock-out” vectors in which a heterologous gene to be expressed in a host is inserted into a known locus or site within the host's genome. The “knock-out” vector consists of an expression cassette flanked by nucleic acid sequences homologous to the nucleic acid sequences flanking the site in the locus where the heterologous gene will be inserted. Typically, the flanking nucleic acid sequences are homologous to the nucleic acid sequences flanking an existing gene that is either known to encode a protein that is not essential for the host's survival or a gene whose absence may be complemented. The inserted expression cassette will replace all or part of the gene at the insertion site. The open reading frame (ORF) regions of genes are typically used as insertion loci because the nucleic acid sequences encoding ORFs are usually well-characterized in terms of structure and function. Because untranslated areas of the host genome are not well understood, insertions into those regions may have unpredictable effects on the host which may fatally or adversely affect growth of the host to such an extent as to render the host unsuitable for expressing the heterologous protein.
However, the number of loci within a host genome's coding sequences, or ORFs, known to encode for non-essential proteins may be quite limited. Additionally, once a given integration locus has been used, it is not possible to use that same locus for further integration within the host. Furthermore, because the ‘knock-out’ method of transformation replaces a functional unit within the host genome, there may be significant perturbation of the genome, which will eventually exhibit adverse effects on the host's productivity or viability.
A second method of constructing recombinant hosts typically involves the use of “roll-in vectors” in which an expression cassette may be inserted using a single cross-over at an insertion locus within the host genome. Because only a single site is used for integration, it is possible to use either coding sequence or non-translated sequence within the locus. If a known ORF is used for the insertion site, the “roll-in” vector may also “knock out” expression of the ORF thereby eliminating expression of the encoded protein and, therefore, will have the disadvantages enumerated above for “knock out” vectors. If a roll-in vector is designed to insert an expression cassette into an untranslated sequence locus, it is possible that minimal adverse effects on the host will be observed. However, it is also possible that the untranslated sequence locus may entail an essential function, in which case unpredictable adverse effects may occur.
The use of roll-in vectors has further disadvantages. In particular, because the roll-in vector relies upon the occurrence of only a single recombination event to integrate into the genome, it is relatively unstable, and the vector may be eliminated from the cell by further recombinant events. Also, the roll-in vector results in an unpredictable gene copy number because multiple insertions may occur either at the same or different insertion sites.
In order to extend the engineering of recombinant expression systems, and to further the development of novel expression systems such as the use of lower eukaryotic hosts to express mammalian proteins with human-like glycosylation, it is necessary to design improved methods and materials to extend the skilled artisan's ability to accomplish complex goals, such as integrating multiple genetic units into a host, with minimal disturbance of the genome of the host organism.